Toll-like receptors (TLRs) have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unclear. Elucidating the mechanisms whereby the lung maintains structural integrity in the face of complex external and internal environments is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, occurring to a certain extent in the normal aging process but pathologically accelerated with chronic exposure to pollutants or cigarette smoke. The fact that only 10 to 20% of smokers develop clinically significant emphysema indicates that other factors are important to its pathogenesis. Recent human studies indicate that depressed TLR4 function is correlated with the severity of COPD, which lends human relevance to our finding that TLR4-/- mice develop spontaneous emphysema after 2 months of age, when lung development is complete. We found that TLR4 deficiency induces a novel NADPH oxidase (Nox), Nox3, and excessive oxidant generation, also a feature of human emphysema. Treatment of TLR4-/- mice or lung endothelial cells with Nox inhibitors or Nox3 siRNA reversed the phenotype. We generated inducible, lung-targeted Nox3 transgenic mice that develop emphysema and also detected elevated Nox3 levels in patients with COPD, indicating an important role for Nox3 in the pathogenesis of emphysema in vivo. In order to investigate the cell type responsible for these TLR4-mediated responses, we generated bone marrow chimeras and found that TLR4 expression on non-hematopoietic cells is required to maintain normal lung architecture. Reconstitution of TLR4 to the airway epithelium only partially prevented emphysema in TLR4-/- and did not suppress Nox3 expression or oxidant generation, indicating that TLR4 in an alternative structural cell is also involved in regulating lung oxidants. Our data using primary lung endothelial cells point to an important role for endothelial TLR4, which we will explore in this proposal. Our hypothesis is that endothelial TLR4 maintains lung integrity by suppressing Nox3-mediated oxidant generation. We will test this hypothesis in the following Aims: 1) Determine the specific contribution of endothelial TLR4 in preventing oxidant-mediated emphysema in vivo, 2) Elucidate the mechanisms whereby TLR4 inhibits Nox3 gene expression in endothelial cells, and 3) Characterize the TLR4 ligand(s) involved in inhibiting Nox3 expression and preventing oxidant-mediated emphysema in vivo and in lung endothelial cells. PUBLIC HEALTH RELEVANCE: Emphysema is a life-threatening destruction of the lungs and can progress even after cessation of identifiable precipitants such as cigarette smoking;however, effective therapies do not yet exist for this devastating disease. Therefore, it is imperative that we understand ways in which the lung maintains its normal structure and function. Our proposed studies will expand upon our recent findings of novel pathways involved in emphysema and our results will identify new therapeutic targets.